U.S. patent application number 17/615285 was filed with the patent office on 2022-08-11 for semiconductor based biosensor utilizing the field effect of a novel complex comprising a charged nanoparticle.
The applicant listed for this patent is LIFE SCIENCE INKUBATOR SACHSEN GMBH & CO. KG. Invention is credited to Norman GERSTNER, Tom STUECKEMANN.
Application Number | 20220252583 17/615285 |
Document ID | / |
Family ID | 1000006359082 |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220252583 |
Kind Code |
A1 |
STUECKEMANN; Tom ; et
al. |
August 11, 2022 |
SEMICONDUCTOR BASED BIOSENSOR UTILIZING THE FIELD EFFECT OF A NOVEL
COMPLEX COMPRISING A CHARGED NANOPARTICLE
Abstract
The present invention relates to a biosensor for detecting
analytes comprising a bio-sensing surface which comprises a field
effect transistor and a first binding molecule which is bonded to
the surface of the field effect transistor. Furthermore, the
biosensor comprises a complex comprising second binding molecules
which are conjugated to charged nanoparticles by linker molecules,
wherein at least one second binding molecule conjugated to a
charged nanoparticle interacts with the first binding molecule
wherein the charged nanoparticle is configured to apply a field
effect on the field effect transistor. Moreover, the present
invention provides a method of detecting an analyte by a
biosensor.
Inventors: |
STUECKEMANN; Tom; (Dresden,
DE) ; GERSTNER; Norman; (Dresden, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE SCIENCE INKUBATOR SACHSEN GMBH & CO. KG |
Dresden |
|
DE |
|
|
Family ID: |
1000006359082 |
Appl. No.: |
17/615285 |
Filed: |
June 2, 2020 |
PCT Filed: |
June 2, 2020 |
PCT NO: |
PCT/EP2020/065163 |
371 Date: |
November 30, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/542 20130101;
G01N 33/587 20130101; G01N 33/5438 20130101; G01N 27/4145 20130101;
G01N 2333/4737 20130101; G01N 27/4146 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/542 20060101 G01N033/542; G01N 33/58 20060101
G01N033/58; G01N 27/414 20060101 G01N027/414 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2019 |
EP |
19177628.5 |
Claims
1. A biosensor for detecting analytes comprising a bio-sensing
surface which comprises a field effect transistor and a first
binding molecule which is bonded to the surface of the field effect
transistor; and a complex comprising second binding molecules which
bind to the first binding molecule and which are conjugated to
charged nanoparticles by linker molecules, wherein, at least one
second binding molecule is conjugated to one charged nanoparticle;
the at least one second binding molecule conjugated to a charged
nanoparticle interacts with the first binding molecule wherein the
charged nanoparticle is configured to apply a field effect on the
field effect transistor; the affinity of the at least one second
binding molecule to the first binding molecule is adaptable such
that the first binding molecule releases the complex comprising the
at least one second binding molecule in presence of the analyte;
and the field effect transistor is configured such that the current
measured in dependence of a voltage applied to said field effect
transistor is changed due to displacement of the complex comprising
the at least one second binding molecule from the first binding
molecule by the analyte.
2. A biosensor according to claim 1, wherein the complex comprises
a charged nanoparticle selected from a group consisting of metallic
nanoparticles, semiconductor nanoparticles, quantum dots or
non-metallic nanoparticles, wherein the nanoparticles are charged
to carry a positive or negative charge; at least one linker
molecule selected from a group consisting of a bond, alkyl,
polyethylene glycol (PEG), polyamide, peptide, carbohydrate,
oligonucleotide or polynucleotide; and at least one second binding
molecule selected from a group consisting of proteins, peptides,
nucleic acids or synthetic components.
3. A biosensor according to claim 1, wherein one second binding
molecule is conjugated to one charged nanoparticle.
4. A biosensor according to claim 1, wherein the affinity of the at
least one second binding molecule to the first binding molecule is
less compared to the affinity of the analyte to the first binding
molecule.
5. A biosensor according to claim 1, wherein the first binding
molecule is selected from proteins, peptides, nucleic acids or
antibodies and fragments thereof.
6. A biosensor according to claim 1, wherein the nanoparticle is a
metallic nanoparticle and is selected from a group consisting of
gold, silver, titanium and platinum, or the nanoparticles are
magnetic metallic nanoparticles selected from Fe.sub.3O.sub.4, or
wherein the nanoparticle is a semiconductor nanoparticle selected
from a group consisting of SiO.sub.2 or the nanoparticle is a
quantum dot selected from a group consisting of CdSe/CdS, CdSe/ZnS,
InAs/CdSe, ZnO/MgO, CdS/HgS, CdS/CdSe, ZnSe/CdSe, MgO/ZnO,
ZnTe/CdSe, CdTe/CdSe and CdS/ZnSe.
7. A biosensor according to claim 6, wherein the nanoparticle is
functionalized with SH-PEG-COOH to carry a negative charge; or
wherein the nanoparticle is functionalized with SH-PEG-NFh to carry
a positive charge.
8. A biosensor according to claim 1, wherein additional charged
compounds are conjugated to the charged nanoparticle.
9. A biosensor according to claim 8, wherein charged compounds
selected from charged peptides or nucleic acids are conjugated to
the charged nanoparticle.
10. A biosensor according to claim 1, wherein Cys-negative charged
peptides or Cys-positive charged peptides are conjugated to the
charged nanoparticle.
11. A method of detecting an analyte with a biosensor wherein the
method comprises the steps of i. providing a biosensor with a
bio-sensing surface which comprises a field effect transistor and a
first binding molecule which is bonded to the surface of the field
effect transistor; ii. selecting a second binding molecule with a
lower affinity to the first binding molecule compared to the
analyte; iii. conjugating the second binding molecules to charged
nanoparticles via linker molecules; iv. bonding the second binding
molecules, which are conjugated to charged nanoparticles via linker
molecules, to the first binding molecule of the biosensing surface;
v. measuring the field effect of the charged nanoparticles to the
field effect transistor by measuring the current in dependence of a
voltage applied to the field effect transistor; vi. contacting the
analyte with the bio-sensing surface and the charged nanoparticles
which are conjugated to second binding molecules; vii. measuring
the change of the field effect acting on the field effect
transistor in presence of the analyte by measuring the current in
dependence of a voltage applied to the field effect transistor,
wherein the second binding molecules conjugated to charged
nanoparticles are partially or completely displaced by analytes due
to the higher affinity of the analytes to the first binding
molecules, thereby changing the field effect acting on the field
effect transistor.
12. The method of detecting an analyte by a biosensor according to
claim 11, wherein the concentration of the analyte is calculated by
the change of the current in dependence of a voltage applied to the
field effect transistor.
13. The method according to claim 11, wherein the second binding
molecules and the charged nanoparticles are conjugated by a
standard two step procedure.
14. The method according to claim 11, wherein the analyte is
present in a physiological solution selected from blood, serum,
saliva, urine, stool or plasma.
15. The method according to claim 11, wherein the field effect of
the analyte acting on a field effect transistor is lower compared
to the field effect of the second binding molecule conjugated to a
charged nanoparticle, wherein the field effect of the analyte and
of the charged nanoparticle on the field effect transistor is
determined by measuring the current in dependence of a voltage
applied to the field effect transistor.
16. The biosensor according to claim 1, wherein said biosensor is
configured to detect an analyte which is present in a physiological
solution selected from blood, serum, saliva, urine stool or
plasma.
17. The biosensor according to claim 1, wherein the field effect of
the analyte acting on a field effect transistor is lower compared
to the field effect of the second binding molecule conjugated to a
charged nanoparticle, wherein the field effect of the analyte and
of the charged nanoparticle on the field effect transistor is
determined by measuring the current in dependence of a voltage
applied to the field effect transistor.
Description
[0001] The present invention relates to a biosensor for detecting
analytes comprising a bio-sensing surface which comprises a field
effect transistor and a first binding molecule which is bonded to
the surface of the field effect transistor. Furthermore, the
biosensor comprises a complex comprising second binding molecules
which are conjugated to charged nanoparticles by linker molecules,
wherein at least one second binding molecule conjugated to a
charged nanoparticle interacts with the first binding molecule
wherein the charged nanoparticle is configured to apply a field
effect on the field effect transistor. Moreover, the present
invention provides a method of detecting an analyte by a
biosensor.
BACKGROUND OF THE INVENTION
[0002] Biosensors include a biological receptor linked on an
electrical transducer in such a way that biological interactions
are translated into electrical signals.sup.1,2. Semiconductor based
Field Effect Transistors (FETs) have received significant attention
as highly sensitive transducers suitable for building fast and
inexpensive diagnostic devices.sup.3-12. However the ability of
FETs to measure all relevant analytes (biomarkers) with a great
sensitivity in physiological solutions like blood, serum or plasma
remains challenging due to the phenomenon of charge screening or
Debye screening in high salt concentrations.sup.3,6,7,13.
[0003] Several attempts to increase the sensitivity of FETs in
physiological solutions have been made.sup.3,5-7,14,15. The
explored strategies can be categorized in four groups:
[0004] 1. Material
[0005] Different semiconductor materials e.g. Carbon Nanotubes
(CNTs).sup.6, Graphen.sup.3, Si-Nanowires.sup.14 and many more have
been used.
[0006] 2. Modifications
[0007] Several sensor surface modifications have been described
e.g. poly ethylene glycol (PEG) has been used in high ionic
strength solutions to increase the sensitivity of CNTs and
graphene.sup.3,6 or the US Patent Application (US2006/0205013) has
used Pyrene groups on the sensor surface to induce charges of
nuclear acids.sup.15.
[0008] 3. Passivation
[0009] Several sensor surface passivation steps, to reduce current
leakage from the source electrode to the drain electrode, through
the applied sample, bypassing the semiconductor material, have been
published. For CNT-FETs the US Patent (US 2012/0073992) has
described polymers like Teflon, polydimethylsiloxane (PDMS),
polymethylmethacrylate (PMMA), silicon dioxide (SiO2), or silicon
nitride (SiN), whereas Filipiak et. al applied SU-8 2005, to reduce
leakage current.sup.6,8. For Si based transistors anti-adhesion
protective molecules like poly ethylene glycol (PEG) terminated
self-assembled monolayers, or benzene terminated self-assembled
monolayers, have been described.sup.14.
[0010] 4. Capture Molecule
[0011] Since the electric field strength falls as the inverse
square of the distance between target and nanomaterial surface, the
size of the capture molecule is important. Therefore different
capture molecules like antibodies, antibody fragments, enzymes,
nanobodies, aptamers or nucleic acids have been coupled on a
diverse set of different semiconductor
materials.sup.2,3,6,13,16,17.
[0012] Despite of all those FET Biosensors optimization, the number
of measurable analytes is very small. This means that some
biomarkers can be detected very well by FET Biosensors whereas
others can't. The main reasons behind this phenomena are the
heterogenic physical/chemical properties among the individual
biomarkers. Especially important for generating a field effect on
the semiconductor material is the relative charge density of an
analyte. The relative charge density is calculated by dividing [the
number of surface charges at neutral pH] through [the diameter of
the biomarker in nm]. Thereby small, but highly charged analytes
like 21 mer miRNAs (21 charges/1 nm=21) generate a strong field
effect and can be much easier detected compared to large, uncharged
molecules like Interleukin 6 (5 charges/3 nm=1.67. Consequently,
the size and the charge of an analyte are critical in order to
generate a field effect. However, since biomarkers are selected for
their clinical indication instead of their relative charge density,
it is extremely challenging to build a biosensor, which can measure
a range of diverse analytes. Therefore, no FET biosensor has been
reported, which is suitable to measure most of the relevant
biomarkers under physiological conditions.
[0013] It is the task of the present invention to provide a
biosensor for detecting analytes, which overcomes the current
limitations of semiconductor based biosensors.
[0014] This task is solved by the present invention by providing a
biosensor for detecting analytes, which comprises [0015] a
bio-sensing surface, wherein the bio-sensing surface comprises a
field effect transistor and a first binding molecule which is
bonded to the surface of the field effect transistor. [0016] and a
complex, which comprises second binding molecules which are
conjugated to charged nanoparticles by linker molecules; wherein at
least one second binding molecule is conjugated to one charged
nanoparticle and wherein the at least one second binding molecule
conjugated to a charged nanoparticle interacts with the first
binding molecule wherein the charged nanoparticle is configured to
apply a field effect on the field effect transistor. Furthermore,
the affinity of the second binding molecule to the first binding
molecule is adaptable such that the first binding molecule releases
the complex comprising the second binding molecule in presence of
the analyte and the current measured in dependence of a voltage
applied to the field effect transistor is changed due to
displacement of the complex comprising the second binding molecule
by the analyte.
[0017] In the overall context of the invention, the wording `low
affinity molecule` or `second binding molecule` means a molecule
contained in the complex of the invention which is able to bind to
the first binding molecule of the bio-sensing surface. Further
characteristics of the low affinity molecules or second binding
molecule are described below.
[0018] Furthermore, the present invention provides a method of
detecting an analyte by a biosensor, said method comprising a
biosensor, wherein said biosensor comprises a bio-sensing surface
which comprises a field effect transistor and a first binding
molecule which is bonded to the surface of the field effect
transistor. Furthermore, the biosensor comprises a complex
comprising second binding molecules which are conjugated to charged
nanoparticles by linker molecules. According to the invention the
method comprises the following steps [0019] i. selecting a second
binding molecule with a lower affinity to the first binding
molecule compared to the analyte; [0020] ii. conjugating the second
binding molecules to charged nanoparticles; [0021] iii. bonding the
second binding molecules which are conjugated to charged
nanoparticles to the bio-sensing surface; [0022] iv. measuring the
field effect of the charged nanoparticles to the field effect
transistor by measuring the current in dependence of a voltage
applied to the field effect transistor; [0023] v. contacting the
analyte with the bio sensing surface and the charged nanoparticles
which are conjugated to second binding molecules; [0024] vi.
measuring the change of the field effect acting on the field effect
transistor by measuring the current in dependence of a voltage
applied to the field effect transistor, wherein the second binding
molecules conjugated to charged nanoparticles are partially or
completely displaced by analytes due to the higher affinity of the
analytes to the first binding molecules, thereby changing the field
effect acting on the field effect transistor.
[0025] In a preferred embodiment, the invention provides a method
of detecting an analyte with a biosensor, wherein the method
comprises the steps of [0026] i. providing a biosensor with a
bio-sensing surface which comprises a field effect transistor and a
first binding molecule which is bonded to the surface of the field
effect transistor; [0027] ii. selecting a second binding molecule
with a lower affinity to the first binding molecule compared to the
analyte; [0028] iii. conjugating the second binding molecules to
charged nanoparticles via linker molecules; [0029] iv. bonding the
second binding molecules, which are conjugated to charged
nanoparticles via linker molecules, to the first binding molecule
of the bio-sensing surface; [0030] v. measuring the field effect of
the charged nanoparticles to the field effect transistor by
measuring the current in dependence of a voltage applied to the
field effect transistor; [0031] vi. contacting the analyte with the
bio-sensing surface and the charged nanoparticles which are
conjugated to second binding molecules; [0032] vii. measuring the
change of the field effect acting on the field effect transistor in
presence of the analyte by measuring the current in dependence of a
voltage applied to the field effect transistor, wherein the second
binding molecules conjugated to charged nanoparticles are partially
or completely displaced by analytes due to the higher affinity of
the analytes to the first binding molecules, thereby changing the
field effect acting on the field effect transistor.
[0033] According to the invention the semiconductor surface is
modified with a binding molecule with which the second binding
molecule interacts in such a way that the charged nanoparticles can
apply a field effect on the semiconductor material. The affinity of
the second binding molecules is selected in such a way that the
first binding molecule can release the second binding molecule in
the presence of an analyte but does not significantly release the
second binding molecule in absents of an analyte. Because the
analyte triggers the release of the second binding molecule the
conjugated charge carrying nanoparticle cannot longer apply a field
effect on the semiconductor material. Thereby the applied field
effect on the semiconductor material can be directly correlated to
the analyte concentration applied to the sensor and can be
electrically read out by the current measured in dependence of a
voltage applied to the field effect transistor.
[0034] The biosensor and method of the invention are therefore
based on the replacement or displacement of complexes comprising
conjugates of second binding or low affinity molecules and
nanoparticles from a first binding molecule as described above by
analytes with higher affinity to the first binding molecule and the
resulting changes in the field effect by said replacement or
displacement, which can be measured with high sensitivity. Several
attempts of optimizing field effect transistor-based biosensors
using conjugates of biomolecules and nanoparticles have been
made'', but the displacement approach of the present invention has
not been used so far.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The biosensor according to the invention is based on a
bio-sensing surface and a complex. The bio-sensing surface
comprises a field effect transistor and a first binding molecule
which is bonded to the surface of the field effect transistor.
[0036] In one embodiment of the invention the first binding
molecule is selected from proteins, peptides, nucleic acids,
antibodies and fragments thereof including monoclonal antibodies,
humanized forms of non-human antibodies, single-chain Fv or sFv
antibody fragments, diabodies or isolated antibodies, preferably
the first binding molecule is selected from antibodies and
fragments thereof.
[0037] The term "antibody" is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments
so long as they exhibit the desired biological activity. The
antibody may be an IgM, IgG (e.g. IgG1, IgG2, IgG3 or IgG4), IgD,
IgA or IgE, for example.
[0038] "Antibody fragments" comprise a portion of an intact
antibody, generally the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments: diabodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0039] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e. the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to "polyclonal antibody"
preparations which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies can
frequently be advantageous in that they are synthesized by the
hybridoma culture, uncontaminated by other immunoglobulins. The
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by generally well known recombinant DNA
methods. The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example.
[0040] The monoclonal antibodies herein specifically include
chimeric antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity.
[0041] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain a minimal sequence
derived from a non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementarity-determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences.
[0042] These modifications are made to further refine and optimize
antibody performance. In general, the humanized antibody will
comprise substantially all or at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321:522-525 (1986), Reichmann et
al, Nature. 332:323-329 (1988): and Presta, Curr. Op. Struct.
Biel., 2:593-596 (1992). The humanized antibody includes a
Primatized.TM. antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by immunizing macaque
monkeys with the antigen of interest.
[0043] "Single-chain Fv" or "sFv" antibody fragments comprise the
VH and VL domains of an antibody, wherein these domains are present
in a single polypeptide chain. Generally, the Fv polypeptide
further comprises a polypeptide linker between the VH and VL
domains which enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0044] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VD) in the same polypeptide chain (VH-VD). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in Hollinger et al., Proc. Natl.
Acad. Sol. USA, 90:6444-6448 (1993). An "isolated" antibody is one
which has been identified and separated and/or recovered from a
component of its natural environment. Contaminant components of its
natural environment are materials which would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the antibody will be purified
(1) to greater than 95% by weight of antibody as determined by the
Lowry method, and most preferably more than 99% by weight, (2) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (3) to homogeneity by SDS-PAGE under reducing or nonreducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the antibody in situ within recombinant
cells since at least one component of the antibody's natural
environment will not be present. Ordinarily, however, isolated
antibody will be prepared by at least one purification step.
[0045] The bio-sensing surface further comprises a filed effect
transistor (FET), wherein every field effect transistor known in
the art is suitable to be used in the invention. Especially,
CNT-FET (carbon nanotube based FET) like swCNT-FET (single walled
carbon nanotube based FET) or mwCNT-FET (multi walled carbon
nanotube based FET) furthermore silicon nanowire FETs or any other
nanoscale semiconductor material are suitable. Preferably
swCNT-FETs are used in the invention.
[0046] Further, the invention comprises a complex which comprises
second binding molecules which are conjugated to charged
nanoparticles by linker molecules.
[0047] According to the invention the complex comprises [0048] a
charged nanoparticle selected from a group comprising metallic
nanoparticles, semiconductor nanoparticles, quantum dots or
non-metallic nanoparticles, most preferable from metal
nanoparticles, wherein the nanoparticles are charged to carry a
positive or negative charge; [0049] at least one linker molecule
selected from a group comprising a bond, alkyl, polyethylene glycol
(PEG), polyamide, peptide, carbohydrate, oligonucleotide or
polynucleotide, most preferable from PEG; and [0050] at least one
second binding molecule selected from a group comprising proteins,
peptides, nucleic acids or synthetic components, preferably from
peptides.
[0051] In a preferred embodiment of the invention the charged
nanoparticles are conjugated by a linker molecule to a second
binding molecule, building the following structure:
A-L-B
wherein A is a second binding molecule, L is a linker molecule, and
B is a charged nanoparticle.
[0052] In another embodiment of the invention more than one second
binding molecule is conjugated to a charged nanoparticle. According
to the invention 1 to 100, preferably 1 to 50 second binding
molecules can be conjugated to one charged nanoparticle, wherein
every second binding molecule is conjugated by a linker
molecule.
[0053] In a preferred embodiment one second binding molecule is
conjugated to one charged nanoparticle. In that event,
consequently, one second binding molecule bound to one charged
nanoparticle can be bound to one binding molecule on the
bio-sensing surface following the laws of affinity as described
below. If more than one second binding molecule is bound to one
charged nanoparticle the laws of avidity take hold. Which means
that multivalent bonds of on charged nanoparticle having several
second binding molecules bound to the first binding molecules on
the bio-sensing surface are possible. This could lead to higher
association constants for the second binding molecule and the first
binding molecule compared to the association constant if one second
binding molecule bound to one charged nanoparticle is exclusively
bound to one binding molecule. Nevertheless, surprisingly,
according to the invention it has turned out that avidity effects
can be neglected if the ratio of second binding molecule to charged
nanoparticle is in the range of 1 charged nanoparticle to 1 to 100
second binding molecules.
[0054] The second binding molecule is selected from proteins,
peptides, nucleic acids or synthetic components, preferably from
peptides.
[0055] In biochemistry, affinity is a measure of the tendency of
molecules to bind to other molecules. The association constant can
be used to quantify the affinity between two binding partners,
where the higher the affinity, the greater the association
constant. Using the example of the formation of a complex ES from
the binding partners E and S
E + S .rarw. k .times. .times. 1 ' .fwdarw. k .times. .times. 1
.function. [ ES ] , ( 1 ) ##EQU00001##
[0056] The association constant K.sub.a is defined as
K a = [ E .times. S ] [ E ] * [ S ] , ( 2 ) K a = k .times. 1 k
.times. .times. 1 ' , respectively . ( 3 ) ##EQU00002##
[0057] k.sub.1 and k.sub.1' are the rate constants for the
association of E and S and the dissociation of the complex ES,
respectively. Analog consideration can be carried out for the
dissociation constant, which is the reciprocal of the association
constant.
[0058] Since it turned out that avidity effects can be neglected
for ratios of second binding molecules to charged nanoparticles as
used according to the invention, even in case of multivalent bonds
the association constant can be approximated according to the above
definition. Meaning that for the purpose of the present invention
the affinity constant for molecules with multivalent bonds can be
approximated by the affinity constant for molecules with a single
bond.
[0059] Basically, the association constant K.sub.a of the second
binding molecule and the first binding molecule is smaller compared
to the association constant of the analyte and the first binding
molecule. What is expressed in the following by the wording, the
second binding molecule has a lower affinity than the analyte for
binding to the first binding molecule. Furthermore, a lower
affinity of the second binding molecule to the first binding
molecule compared to the affinity of the analyte to the first
binding molecule results in displacement of the second binding
molecule from the binding site of the first binding molecule in
presence of the analyte by the analyte.
[0060] Therefore, in a preferred embodiment of the invention the
affinity of the second binding molecule to the first binding
molecule is smaller compared to the affinity of the analyte to the
first binding molecule.
[0061] It is crucial for the present invention that the affinity of
the second binding molecule to the first binding molecule of the
bio-sensing surface is less compared to the affinity of the analyte
to the first binding molecule of the bio-sensing surface. This
property guarantees that the complex is released in the presence of
the analyte. Moreover, the affinity of the second binding molecule
must be in a range that the second binding molecule is bound to the
first binding molecule of the bio-sensing surface in absence of the
analyte, so that the charged nanoparticle comprised in the complex
can apply a field effect on the field effect transistor of the
bio-sensing surface.
[0062] The second binding molecule may be further modified in such
a way that the affinity for binding to the first binding molecule
is altered, in particular reduced, compared to the intact analyte.
Which means K.sub.a of second binding molecule and first binding
molecule is reduced compared to K.sub.a of analyte and first
binding molecule. Thereby the capability of the analyte to displace
the complex captured at the binding site of the first binding
molecule is improved. Altering, i.e. lowering of the affinity of
the molecule captured at the antibody binding site may be achieved
by point mutation, chemical modification by, e.g. biotinylation,
glycosylation or any other method known in art.
[0063] In one embodiment of the invention the affinity of the
second binding molecule is altered by point mutation, chemical
modification by, e.g. biotinylation, glycosylation or any other
method known in art.
[0064] In a preferred embodiment, the second binding molecule part
of the complex captured at the binding site of the first binding
molecule is a fragment of an antigen. The fragment of an antigen
may be known in the art or is a synthetic peptide, wherein the
amino acid sequence of a synthetic peptide is suitably adapted such
that the binding to the binding site of the first binding molecule
of the invention is facilitated.
[0065] In a more preferred embodiment, such antigen fragment or
synthetic peptide has a chain length of 4 to 22 amino acids, more
preferably of 5 to 15 amino acids, most preferably of 6 to 12 amino
acids.
[0066] A further modification of the affinity of the second binding
molecule to the first binding molecule of the bio-sensing surface
by the above described measures has the advantage that the affinity
of the second binding molecule to the first binding molecule of the
bio-sensing surface can be regulated to be in an optimal range.
Optimal range meaning that the second binding molecule is bound to
the first binding molecule of the bio-sensing surface in absence of
the analyte but is released in presence of the analyte.
[0067] The complex further comprises a linker molecule, which is
selected from a bond, alkyl, polyethylene glycol (PEG), polyamide,
peptide, carbohydrate, oligonucleotide or polynucleotide, most
preferable from PEG.
[0068] The linker molecule is further characterized in that it has
a Maleimide group on its proximal end and a NH.sub.2 group or an
NHS-Ester group or a Sulfo-NHS-Ester group on its distal end.
[0069] Therefore in a preferred embodiment of the invention the
linker molecule has a Maleimide group on its proximal end and a
NH.sub.2 group or an NHS-Ester group or a Sulfo-NHS-Ester group on
its distal end.
[0070] For example suitable linker molecules are selected from a
group comprising Mal-PEG-NH2, Mal-PEG-SulfoNHS, Mal-PEG-NHS.
[0071] PEG is an oligomer or polymer composed of ethylene oxide
monomers with the following monomer structure
(--CH.sub.2--CH.sub.2--O--).sub.n. Because different applications
require different polymer chain lengths, PEGs are prepared by
polymerization of ethylene oxide and are commercially available
over a wide range of molecular weights from 300 g/mol to 10,000,000
g/mol. While PEGS with different molecular weights find use in
different applications, and have different physical properties
(e.g. viscosity) due to chain length effects, their chemical
properties are nearly identical. Different forms of PEG are also
available, depending on the initiator used for the polymerization
process--the most common initiator is a monofunctional methyl ether
PEG, or methoxypoly (ethylene glycol), abbreviated mPEG.
Lower-molecular-weight PEGs are also available as purer oligomers,
referred to as monodisperse, uniform, or discrete.
[0072] PEGS are also available with different geometries: [0073]
Linear PEGs, where the ethylene oxide monomers are bound to each
other in an unbranched polymer chain; [0074] Branched PEGs, which
have three to ten PEG chains emanating from a central core group;
[0075] Star PEGs, which have 10 to 100 PEG chains emanating from a
central core group; and [0076] Comb PEGs, which have multiple PEG
chains normally grafted onto a polymer backbone.
[0077] The numbers that are often included in the names of PEGs
indicate their average molecular weights (e.g. a PEG with n=9 would
have an average molecular weight of approximately 400 daltons, and
would be labeled PEG 400). Most PEGs include molecules with a
distribution of molecular weights (i.e. they are polydisperse). The
size distribution can be characterized statistically by its weight
average molecular weight (Mw) and its number average molecular
weight (Mn), the ratio of which is called the polydispersity index
(Mw/Mn). Mw and Mn can be measured by mass spectrometry.
[0078] PEG is soluble in water, methanol, ethanol, acetonitrile,
benzene, and dichloromethane, and is insoluble in diethyl ether and
hexane.
[0079] In a preferred embodiment, the linker of the invention
comprises a linear PEG. Using linear PEGS has the advantage that
they are cheap and possess a narrower molecular weight
distribution.
[0080] When linear PEG is used to form the linker of the conjugate
of the invention, it has suitably a molecular weight in the range
of 40 Da to 10,000 Da, preferably in the range of 200 Da to 6,000,
more preferably in the range of 400 Da to 4,000 Da, most preferably
in the range of 1,000 Da to 3,400 Da.
[0081] Furthermore, according to the invention charged
nanoparticles are comprised in the complex, which are used as
charge carrying objects, able to apply a proper field effect on the
field effect transistor of the bio-sensing surface.
[0082] Charged nanoparticles are selected from a group comprising
metallic nanoparticles, semiconductor nanoparticles, quantum dots
or non-metallic nanoparticles, most preferable from metal
nanoparticles, wherein the nanoparticles are charged to carry a
positive or a negative charge. Suitable non-metallic nanoparticles
are for example nanoparticles comprising carbides or nitrides, like
aluminum nitride, boron nitride, boron carbide, silicon carbide,
silicon nitride, titanium carbide, titanium nitride, tungsten
carbide, tungsten nitride or zirconium carbide.
[0083] Furthermore, suitable non-metallic nanoparticles are for
example oxides comprising Antimony(III) oxide, Antimon Tin Oxide
(ATO), Aluminium Zinc Oxide (AZO), Barium titanate (BaTiO3),
Bismuth(III) oxide (Bi2O3), Cerium(IV) oxide (CeO2), Chromium(III)
oxide (Cr2O3), Cobalt(II, III) oxide (Co3O4), Copper(II) oxide
(CuO), Dysprosium(III) oxide (Dy2O3), Erbium(III) oxide (Er2O3),
Europium(III) oxide (Eu2O3), Gadolinium(III) oxide (Gd2O3),
Hafnium(IV) oxide (HfO2), Indium(III) oxide (In2O3), Iron(II, III)
oxide (Fe3O4), Indium Tin Oxide (ITO), Lanthanum(III) oxide
(La2O3), Magnesium(II) oxide (MgO), Neodymium(III) oxide (Nd2O3),
Nickel(II) oxide (NiO), Samarium(III) oxide (Sm2O3), Silicon(IV)
oxide (SiO.sub.2), Strontium titanate (SrTiO3), Tin(IV) oxide
(SnO2), Titanium(IV) oxide (TiO2), Yttrium(III) oxide (Y2O3), Zinc
oxide (ZnO), Zirconium(IV) oxide (ZrO2), .alpha.-Aluminium oxide
(Al.sub.2O.sub.3), .alpha.-Iron(III) oxide (Fe2O3),
.gamma.-Aluminium oxide (Al.sub.2O.sub.3) or .gamma.-Iron(III)
oxide (Fe2O3).
[0084] Charged nanoparticles used in the complex of the invention
have a molecular size in the range of 1-100 nm, preferably in the
range of 5-50 nm, more preferably in the range of 5-40 nm, most
preferably in the range of 5-20 nm.
[0085] In one embodiment of the invention the nanoparticles are
metal nanoparticles which are selected from a group comprising
gold, silver, titanium and platinum or the nanoparticles are
magnetic metallic nanoparticles selected from Fe3O4.
[0086] However, metal nanoparticles, especially gold nanoparticles
have been already described as signaling tools in many
analytic/diagnostic applications. Different sizes and shapes of
gold nanoparticles are for example used in lateral flow assays, the
latter for generating duo-colored lateral flow tests.sup.18. Also
peptide functionalized gold nanoparticles have been described in a
variety of applications. In the detection of metal ions, several
studies have appeared in the literature.sup.19-22. Whereby all
described methods are based on colorimetric measurements of the
spectral shift triggered by gold particle aggregation. The same
physical measurement principle has been successfully applied to
measure matrix metallo-proteinase matrilysin (MMP-7).sup.23,
neurofenin 3 (ngn3).sup.24, bluetongue virus (BTV)-specific
antibodies.sup.25 or blood coagulation factor XIII.sup.26. Even a
cardiac Troponin-I assay, based on peptide functionalized gold
nanorods, has been described.sup.27. In contrast to the described
assays the presented invention does not use the combination of
optical and electronic properties of gold nanoparticles as
signaling system. Instead this invention uses functionalized gold
nanoparticles as charge carrying object, which are able to apply a
field effect on semiconductor materials as signaling system.
Accordingly, the invention uses the physical properties of the gold
nanoparticles in a completely different and novel way.
[0087] In another embodiment of the invention the nanoparticles are
semiconductor nanoparticles selected from a group comprising
SiO.sub.2.
[0088] In a further embodiment of the invention the nanoparticles
are quantum dots selected from a group comprising CdSe/CdS,
CdSe/ZnS, InAs/CdSe, ZnO/MgO, CdS/HgS, CdS/CdSe, ZnSe/CdSe,
MgO/ZnO, ZnTe/CdSe, CdTe/CdSe and CdS/ZnSe.
[0089] In a most preferred embodiment of the invention the
nanoparticles are selected from gold or Fe.sub.3O.sub.4
nanoparticles.
[0090] In a preferred embodiment of the invention the nanoparticles
are charged to carry a positive or a negative charge. This is done
by functionalizing the nanoparticles with SH-PEG-COOH or
SH-PEG-NH.sub.2. A nanoparticle functionalized with a SH-PEG-COOH
group is charged to carry a negative charge, while a nanoparticle
functionalized with a SH-PEG-NH.sub.2 group is charged to carry a
positive charge.
[0091] In a further preferred embodiment the nanoparticles are
functionalized with SH-PEG-COOH groups to carry a negative charge
or with SH-PEG-NH.sub.2 to carry a positive charge.
[0092] The nanoparticles may be further modified with additional
peptides which are characterized in that the peptide sequence
represents polar and un-polar amino acids whereby the polar amino
acids are homogeneously (only positively or negatively charged
amino acids) in their charge. The peptides are further
characterized in that they are between 4 and 25 amino acids long,
whereby negatively charged peptides are exclusively coupled to
SH-PEG-COOH functionalized nanoparticles, and whereas positively
charged peptides are exclusively coupled to SH-PEG-NH.sub.2
functionalized nanoparticles. Suitable peptides comprise a cysteine
residue, for example CLDDD-OH or RRRLC-amid peptides are
usable.
[0093] In one embodiment of the invention additional charged
compounds are conjugated to the charged nanoparticles. Suitable
charged compounds are selected from a group comprising charged
peptides, nucleic acids like DNA and RNA, and are conjugated to the
charged nanoparticles.
[0094] In a preferred embodiment of the invention the charged
peptides additionally conjugated to the charged nanoparticles are
between 4 and 25 amino acids long, preferably between 4 and 20
amino acids long.
[0095] In a preferred embodiment of the invention Cys-negative
charged peptides or Cys-positive charged peptides are conjugated to
the charged nanoparticles.
[0096] Therefore in one embodiment of the invention a CLDDD-OH is
conjugated to a COOH functionalized nanoparticle.
[0097] In a further embodiment of the invention a RRRLC-amid is
conjugated to a NH.sub.2 functionalized nanoparticle.
[0098] Furthermore nucleic acids like DNA and RNA are suitable to
be additionally conjugated to the charged nanoparticle, since their
phosphate backbone is charged. Due to the charged phosphate
backbone any sequence of DNA or RNA is suitable.
[0099] In one embodiment of the invention DNA or RNA is conjugated
to the charged nanoparticles.
[0100] Modifying the charged nanoparticles with additional charged
compounds as described above has the advantage that the field
effect of the charged nanoparticle and therefore of the complex on
the field effect transistor of the bio-sensing surface can be
increased compared to the field effect of charged nanoparticles
without modification. Therefore, the measurable difference between
the field effect of the compound and the analyte to the field
effect on the field effect transistor of the bio-sensing surface is
increased as well.
[0101] Furthermore, a method of detecting an analyte by a biosensor
is provided. Said method comprises a biosensor, wherein said
biosensor comprises a bio-sensing surface which comprises a field
effect transistor and a first binding molecule which is bonded to
the surface of the field effect transistor. Furthermore the
biosensor comprises a complex comprising second binding molecules
which are conjugated to charged nanoparticles by linker
molecules.
[0102] In a preferred embodiment the bio-sensing surface and the
complex of the biosensor have the same features as described above
for the biosensor according to the invention. In a further
preferred embodiment the biosensor according to the invention is
used in the method.
[0103] The method according to the invention comprises the
following steps: [0104] i. selecting a second binding molecule with
a lower affinity to the first binding molecule compared to the
analyte; [0105] ii. conjugating the second binding molecules to
charged nanoparticles; [0106] iii. bonding the second binding
molecules which are conjugated to charged nanoparticles to the
bio-sensing surface; [0107] iv. measuring the field effect of the
charged nanoparticles to the field effect transistor by measuring
the current in dependence of a voltage applied to the field effect
transistor; [0108] v. contacting the analyte with the bio-sensing
surface and the charged nanoparticles which are conjugated to
second binding molecules; [0109] vi. measuring the change of the
field effect acting on the field effect transistor by measuring the
current in dependence of a voltage applied to the field effect
transistor, wherein the second binding molecules conjugated to
charged nanoparticles are partially or completely displaced by
analytes due to the higher affinity of the analytes to the first
binding molecules, thereby changing the field effect acting on the
field effect transistor.
[0110] In step i) a second binding molecule is selected which is
suitable for the measurement requirements. Therefore a second
binding molecule is chosen with a lower affinity to the first
binding molecule of the bio-sensing surface compared to the
analyte. Which means the association constant K.sub.a of the second
binding molecule and the first binding molecule is less compared to
the association constant of the analyte and the first binding
molecule.
[0111] In step ii) the second binding molecules are conjugated to
charged nanoparticles. In a preferred embodiment of the invention
the second binding molecules and the charged nanoparticles are
conjugated by a standard two step procedure.
[0112] Prior the conjugation of the second binding molecules with
the charged nanoparticles, the nanoparticles are functionalized
with carboxyl (COOH) or amino (NH.sub.2) groups to carry a positive
or a negative charge. The functionalization of metal nanoparticles
is performed by the metal-thiol reaction using either SH-PEG-COOH
or SH-PEG-NH2 heterobifunctional reagents with a molecular weight
of 100-10,000 Da, preferable with 200-5,000 Da, more preferable
with 300-3,000 Da, most preferable with 400-1,000 Da.
[0113] Heterobifunctional reagents can also be used to link
nanoparticles to peptides or other molecules in a two- or
three-step process that limits the degree of polymerization often
obtained using homobifunctional crosslinkers. In a typical
conjugation scheme, the nanoparticle is modified with a
heterobifunctional compound using the crosslinker's most reactive
or most labile end. The modified nanoparticle is then purified from
excess reagent by centrifugation or by molecular weight cut-off
columns. Most heterobifunctional linker contain at least one
reactive group that displays extended stability in aqueous
environments, therefore allowing purification of an activated
intermediate before adding the second molecule (e.g peptide) to be
conjugated. For instance, an NHS ester-maleimide heterobifunctional
linker can be used to react with the amine groups of modified
nanoparticles through its NHS ester end (the most labile
functionality), while preserving the activity of its maleimide
functionality. Since the maleimide group has greater stability in
aqueous solution than the NHS ester group, a maleimide-activated
intermediate may be created.
[0114] After a quick purification step, the maleimide end of the
crosslinker can then be used to conjugate to a sulfhydryl
containing molecule (e.g. a peptide via a cysteine residue).
[0115] Such multi-step protocols offer greater control over the
resultant size of the conjugate and the molar ratio of components
within the crosslinked product. The configuration or structure of
the conjugate can be regulated by the degree of initial
modification of the nanoparticle and by adjusting the amount of
peptide added to the final conjugation reaction.
[0116] The third component of all heterobifunctional reagents is
the cross-bridge or spacer that ties the two reactive ends
together. Crosslinkers may be selected based not only on their
reactivities, but also on the length and type of cross-bridge they
possess. Some heterobifunctional families differ solely in the
length of their spacer. The nature of the cross-bridge may also
govern the overall hydrophilicity of the reagent.
[0117] For instance, polyethylene glycol (PEG)-based crossbridges
create hydrophilic reagents that provide water solubility to the
entire heterobifunctional compound. A few crosslinkers contain
peculiar cross-bridge constituents that actually affect the
reactivity of their functional groups. For instance, it is known
that a maleimide group that has an aromatic ring immediately next
to it is less stable to ring opening and loss of activity than a
maleimide that has an aliphatic ring adjacent to it.
[0118] In a preferred embodiment of the invention
heterobifunctional reagents are used to conjugate second binding
molecules and charged metal particles.
[0119] Suitable heterobifunctional reagents are selected from a
group comprising Mal-PEG-NH2, Mal-PEG-NHS, Mal-PEG-SulfoNHS and
similar cross linkers with a molecular weight of 200-8,000 Da,
preferable with 500-5,000 Da, more preferable with 1,000-4,000 Da,
most preferable with 2,000-3,500 Da.
[0120] In a preferred embodiment of the invention Mal-PEG-NH.sub.2
cross linker is used in combination with carboxyl functionalized
nanoparticles.
[0121] In a further preferred embodiment of the invention
Mal-PEG-NHS or Mal-PEG-SulfoNHS or similar cross linkers are used
in combination with amino functionalized nanoparticles.
[0122] Particle size, surface composition, and density directly
affect how a particle behaves in suspension. This in turn affects
coupling protocols, especially in the handling and washing
techniques used for particles during the conjugation process.
Larger particles of micron size will generally settle over time
just in normal gravity. As particle size decreases, however, a
point is reached where a true colloidal suspension may occur,
wherein the particles will not separate, no matter how long they
sit in suspension. This typically happens when particle size gets
to about 100 nm, and Brownian motion causes water molecules to
collide with particles with high enough force-to-mass ratios to
prevent them from settling under gravity. Many dense particles of
less than 100 nm, such as silica, can still be separated from
solution using a bench-top centrifuge; however, as particles
approach the size of biological macromolecules, or around 10 nm, an
ultracentrifuge would be required for separation.
[0123] For example Au nanoparticles down to a size of 15 nm can be
purified using a standard laboratory centrifuge. Smaller Au
nanoparticles down to 10 nm require ultracentrifugation. For even
smaller Au nanoparticles down to 5 nm molecular weight cut-off
columns for particle purification are necessary.
[0124] The charge repulsion effects between particles can be
severely affected by the buffer and salt composition of the
solution they are suspended in. Charges can be eliminated or
neutralized by ionizable groups being protonated or unprotonated or
by the concentration of ions in solution. For instance, lowering
the pH of an aqueous solution below the pKa of the surface
carboxylates will result in them being protonated. With most
particles, especially ones having hydrophobic surfaces, this will
cause particle aggregation due to loss of surface negative charge.
Similarly, a high salt concentration can effectively mask the
charge character of a carboxylated particle by having too many
positively charged ions associated with the surface negative
charges. Most particle types that are stable in suspension due to
like charge repulsion can be made to aggregate if the pH is changed
or the buffer or salt concentration is too high.
[0125] Part of the challenge of successfully working with small
particles is to maintain optimal solution characteristics to keep
the particles dispersed throughout the conjugation process. This
includes all activation, coupling, and washing steps that are used
to conjugate an affinity ligand, like the low-affinity molecule and
subsequently use it in its intended application.
[0126] If individual particles in suspension are considered the
equivalent of discrete molecules, then the molar concentration of a
given particle suspension can be calculated based on the known
particle diameter, density, and the mass of particles present. This
allows particles to be treated similarly to other biomolecules with
respect to determining concentration for conjugation purposes.
However, there are important differences that should be recognized
when working with particles as opposed to working with soluble
macromolecules, like proteins. Since common commercial particles
can vary in size from the molecular range (approximating the size
of an antibody or -10-nm diameter) to a scale 1,000 times larger
(or approaching the size of a cell at 10 .mu.m), a change in
diameter affects the concentration of particles as well as the
effective concentration of surface functional groups present in
suspension. Also potentially affected are the dispersion
characteristics of particles as their size is changed
(Suttiponparnit et al., 2011).
[0127] In general, as particle size decreases, the molar
concentration of particles in a constant volume of solution
increases (for a given mass of particles). For instance, a 1-mg
quantity of 1-.mu.m latex microspheres represents far fewer
particles than a 1-mg amount of 50-nm nanoparticles. Thus, the
effective molar concentration of nanoparticles in solution will be
much greater than the concentration of the same mass of
microparticles (if both are suspended at the same mass quantity and
in same volume of solution). In addition, as the diameter decreases
for a given mass of particles, the ratio of a particle's surface
area to mass increases. This means that the total surface area
available for conjugation on the nanoparticles is much greater than
the total surface area present on the microparticles. If both
particles contain the same functional groups on their surfaces for
coupling affinity ligands (i.e., carboxylates), then for the same
mass of particles the effective concentration of these functional
groups in solution is much greater for the nanoparticles than the
concentration of the same groups in a given solution for the
microparticles (assuming both have about the same surface density
or "parking area" of the carboxylate functional groups).
[0128] Thus, conjugation reactions performed with nanoparticles
should take into account a potentially greater reactivity than the
same reactions performed using microparticles, due to the higher
effective concentration of functional groups present in solution
for the nanoparticles. As particle size decreases and particle
concentrations increase, the available surface area increases, and
the effective concentration of reactive groups increases along with
it.
[0129] Conjugation of the second binding molecules to the charged
nanoparticles is therefore done in the following two-step
procedure.
[0130] Step 1:
[0131] In order to conjugate peptides via a thiol-maleimide
reaction on carboxylated or aminated nanoparticles, the
nanoparticles have to be functionalized with a maleimide group. In
case of SH-PEG-COOH functionalized nanoparticles, the
functionalization is performed by a Mal-PEG-NH2 heterobifunctional
reagents. In order to react the amino (NH2) group of the
heterobifunctional cross linker with the carboxyl groups of the
nanoparticle, an EDC/Sulfo NHS activation reaction is required.
Therefore EDC and Sulfo-NHS are added to the SH-PEG-COOH
functionalized nanoparticles for 15 min at room temperature. After
activation of carboxyl groups the reaction mixture is purified from
excess reagent by molecular weight cut-off columns and transferred
into PBS (phosphate buffered saline) with pH 7.2. In particular,
the activation of carboxylate particles using an EDC/Sulfo-NHS
reaction will temporarily replace the negatively charged
carboxylates with negatively charged sulfonates. The sulfonate
groups on the Sulfo-NHS ester intermediates create a stronger
negative charge on the particle surface than the original
carboxylates. In some cases, the increase in negative charge
repulsion can result in an inability to pellet the particles by
centrifugation after the activation step even if the particles
could be separated by centrifugation before activation. Therefore,
in this invention molecular weight cut-off columns to purify
reaction intermediates of EDC/sulfo-NHS reactions are used.
[0132] Immediately after purification a 10-100 fold molar excesses
of Mal-PEG-NH2 heterobifunctional cross linker is added for 30 min
at room temperature. The excess reagents are purified off by
molecular weight cut-off columns.
[0133] In case nanoparticles functionalized with SH-PEG-NH.sub.2
are used, the conjugation is performed by a Mal-PEG-NHS or
Mal-PEG-Sulfo-NHS or similar heterobifunctional cross linker. The
reaction takes place at a pH of 7.2-7.5 and will be performed for
15 min at room temperature. A pre-activation step is not required.
A 10-100 fold molar excesses of Mal-PEG-NHS or Mal-PEG-Sulfo-NHS
heterobifunctional cross linker is used. The excess reagents are
purified off by molecular weight cut-off columns
[0134] Step 2:
[0135] The purified nanoparticle-PEG-Mal conjugate should be
directly used for the peptide conjugation reaction. The reaction
should take place at a pH between 6.5 and 7.5. Thiol-containing
compounds, such as dithiothreitol (DTT) and beta-mercaptoethanol
(BME), must be excluded from reaction buffers used with maleimides
because they will compete for coupling sites. For example, if DTT
were used to reduce disulfides, to make sulfhydryl groups available
for conjugation, the DTT would have to be thoroughly removed using
a desalting column before initiating the maleimide reaction.
Interestingly, the disulfide-reducing agent TCEP does not contain
thiols and does not have to be removed before reactions involving
maleimide reagents. Excess maleimides can be quenched at the end of
a reaction by adding free thiols. EDTA can be included in the
coupling buffer to chelate stray divalent metals that otherwise
promote oxidation of sulfhydryls (non-reactive). The conjugated
peptides are used in a 10-1,000 fold molar excess. The reaction
takes place at room temperature for 2-12 hours or at 4.degree. C.
for 4-24 hours. The excess reagents are purified off by molecular
weight cut-off columns.
[0136] Furthermore, additional charged compounds may be added to
the charged nanoparticles. This is done for compounds with a
relative charge density greater than 10, for example for charged
peptides, by a competitive reaction between the second binding
molecule and the charged peptide with the maleimide functionalized
charged Au nanoparticles. First the negative charged nanoparticles
react with a NH.sub.2-PEG-Mal linker (I). In a second reaction step
a mixture of the second binding molecule and the charged peptide is
added, in such a way that the charged peptide has a molar excess of
2-200 fold. In case of positively charged Au nanoparticles first
the amino functionalized Au nanoparticles reacts with NHS-PEG-Mal
heterobifunctional linker. In a second step the second binding
molecule and the positive charged peptide is added, in such a way
that the charged peptide has a molar excess of 2-200 fold.
[0137] According to step iii) of the method the second binding
molecules which are conjugated to charged nanoparticles (complex)
are added to the bio-sensing surface. The second binding molecule
binds to the first binding molecule on the surface of the
bio-sensing surface due to the affinity of both binding partners.
The charged nanoparticles conjugated to the second binding
molecules apply a field effect on the field effect transistor of
the bio-sensing surface. Subsequently (step iv), the field effect
is measured by measuring the current flow through the field effect
transistor in dependence of a voltage applied to the field effect
transistor.
[0138] In a next step the analyte is contacted with the bio-sensing
surface. Due to the higher affinity of the analyte to the first
binding molecule of the bio-sensing surface compared to the
affinity of the second binding molecule to the first binding
molecule of the bio-sensing surface, second binding molecules are
partially or completely displaced by analytes due to the higher
affinity of the analytes to the first binding molecules. The
displacement of the second binding molecules by analytes is
directly proportional to the concentration of the analyte. Due to
the displacement of the second binding molecules and therefore also
of the charged nanoparticles the field effect applied on the field
effect transistor is now caused by the analytes.
[0139] In the last step of the method (step vi) according to the
invention the change of the field effect acting on the field effect
transistor is measured by measuring the current in dependence of a
voltage applied to the field effect transistor. Therefore, the
concentration of the analyte can be calculated by the change of the
current in dependence of a voltage applied to the field effect
transistor.
[0140] In one embodiment of the invention the concentration of the
analyte is calculated by the change of the current in dependence of
a voltage applied to the field effect transistor.
[0141] Advantageously, analytes can be detected which apply a low
or even no measurable field effect on a field effect transistor due
to the phenomenon of charge screening or Debye screening because
the analyte is present in a solution with a high salt
concentration. According to the present invention second binding
molecules and consequently also charged nanoparticles are displaced
by the analyte present in a solution, wherein the displacement is
proportional to the concentration of the analyte in the solution.
Due to the displacement the field effect applied on the field
effect transistor decreases with increasing analyte concentration.
Using this mechanism the concentration of the analyte in a solution
can be measured.
[0142] However, also highly charged analytes can be detected by the
present invention. Charged nanoparticles according to the invention
have a relative charge density of approximately 30-150. The
relative charge density of RNA is approximately 21 and of proteins
even lower. Therefore, the field effect applied by the charged
nanoparticles according to the invention is greater compared to the
analyte in each case of interest. Accordingly, the filed effect
applied by the analyte on the field effect transistor is lower
compared to the field effect applied by the charged
nanoparticles.
[0143] In a preferred embodiment of the invention the field effect
of the analyte acting on a field effect transistor is lower
compared to the field effect of the second binding molecule
conjugated to a charged nanoparticle, preferably the field effect
of the analyte acting on a field effect transistor is too low to be
detectable.
[0144] Therefore, advantageously analytes present in solutions with
high salt concentrations are detectable with the present invention.
Especially analytes present in physiological solutions selected
from blood, serum, saliva, stool, urine or plasma are
detectable.
[0145] Accordingly, the invention describes a novel biosensor and a
method comprising a bio-sensing surface and a complex which are
able to detect biomarkers irrespectively of the physical/chemical
properties. Further, universally all semiconductor materials in
combination with different binding molecules can be used in the
invention in combination with all relevant passivation/modification
steps. Thereby, this invention solves a long standing problem for
the entire biosensor and diagnostic field.
[0146] Additionally and in contrast to all published methods and
procedures of the art, in this invention, the interaction between
the nanoparticle and the first binding molecule, modulated by the
conjugated second binding molecule, happens within a very
well-defined affinity. This means that for any given analyte, a
second binding molecule with a corresponding structure (e.g.
peptide sequence) is selected in such a way that the affinity of
the second binding molecule and the first binding molecule is lower
compared to the affinity between the analyte and the first binding
molecule. Thereby, a displacement reaction between the first
binding molecule and the complex and therefore the charged
nanoparticle, takes place as soon the analyte is added.
Consequently, the applied field effect on the field effect
transistor is altered in such a way that a significant measurement
signal can be obtained.
[0147] In the following, the present invention is further described
by 8 figures and 3 examples.
[0148] FIG. 1 illustrates FET biosensors which are state of the
art;
[0149] FIG. 2 (A) illustrates a charged nanoparticle and (B)
illustrates the biosensor according to the invention;
[0150] FIG. 3 illustrates the signals measurable with a field
effect transistor of a complex according to the invention and an
analyte present in a solution with a high salt concentration;
[0151] FIG. 4 (A) illustrates the functionalization of a gold
nanoparticle to carry a negative charge and its conjugation with a
second binding molecule, (B) illustrates the functionalization of a
gold nanoparticle to carry a positive charge and its conjugation
with a second binding molecule;
[0152] FIG. 5 (A) illustrates the functionalization of a negative
charged gold nanoparticle conjugated to a second binding molecule
which is functionalized with additional Cys-negative charged
peptides, (B) illustrates the functionalization of a positive
charged gold nanoparticle conjugated to a second binding molecule
which is functionalized with additional Cys-positive charged
peptides;
[0153] FIG. 6 shows the results of the affinity measurements of
monoclonal mouse IgG1 anti human CRP antibody B08 against the
biomarker CRP (SEQ ID NO: 1) and modified peptide sequences of SEQ
ID NOs: 2 to 6;
[0154] FIGS. 1 (A) and (B) illustrate state of the art FET
biosensors. FIG. 1 illustrates a semiconductor with an antibody
acting as binding molecule on its surface. In FIG. 1 (A) an analyte
is bound to the antibody and applies a measurable field effect on
the field effect transistor. In several cases, especially when the
analyte is present in solutions with high salt concentration, the
analyte is bound to the antibody but no measurable field effect is
applied to the semiconductor by the analyte, which is due to charge
screening effects.
[0155] FIG. 2 (A) illustrates a charged nanoparticle which is
conjugated to a second binding molecule. The second binding
molecule is bound to a binding molecule on the surface of the field
effect transistor, thereby the charged metal particle applies a
field effect on the field effect transistor which is measurable
(FIG. 2 (B)). If an analyte is added to the biosensor according to
the invention the complex comprising the second binding molecule
and the charged nanoparticle is displaced by the analyte on the
binding site of the first binding molecule on the surface of the
field effect transistor. Due to the displacement the field effect
applied on the field effect transistor is altered. In case the
analyte applies a low or even no field effect on the field effect
transistor the measurable field effect on the field effect
transistor is decreased. Since the displacement of the complex by
the analyte is proportional to the concentration of the analyte,
the change of the field effect is a measure for the concentration
of the analyte in the solution. Therefore, especially analytes
present in solutions with a high salt concentration or
physiological solutions like blood, serum, saliva, stool, urine or
plasma are detectable.
[0156] FIG. 3 illustrates the signals measurable with a field
effect transistor of a complex according to the invention and an
analyte present in a solution with a high salt concentration. The
figure illustrates the current measured with a constant voltage of
a field effect transistor in dependence of the time for two
substances S1 and S2. S1 is a biomarker which is present in a high
salt solution applying a week field effect on the field effect
transistor (dashed line). Substance S2 is a complex according to
the invention also present in a high salt solution. As can be seen
a significantly higher current is measured with the field effect
transistor for S2 (solid line).
[0157] In FIG. 4 (A) a gold nanoparticle is functionalized with a
SH-PEG-COOH to carry a negative charge. Subsequently, the negative
charged gold nanoparticle is conjugated to a Cys-peptide in a
two-step procedure. Firstly, the carboxyl groups of the SH-PEG-COOH
functionalized gold nanoparticle are activated by an EDC/NHS
activation reaction. Afterwards the NH2-PEG-MAL heterobifunctional
reagent is added and a maleimide activated negative charged
nanoparticle is obtained. Secondly, a Cys-peptide is added and
conjugated to the charged gold nanoparticle.
[0158] FIG. 4 (B) illustrates the procedure for a gold nanoparticle
which is functionalized with a SH-PEG-NH.sub.2 to carry a positive
charge. Accordingly, the positive charged gold nanoparticle is
conjugated to a Cys-peptide in a two-step procedure. Firstly, the
NHS-PEG-MAL heterobifunctional reagents is added and a maleimide
activated positive charged nanoparticle is obtained. Secondly, a
Cys-peptide is added and conjugated to the charged gold
nanoparticle.
[0159] Further charged compounds can be added to the complex of the
invention. FIG. 5 (A) illustrates the two-step functionalization
reaction of negative charged metal nanoparticles. First
carboxylated metal nanoparticles are activated by EDC/SulfoNHS
reaction and functionalized with an NH.sub.2-PEG-MAL
heterobifunctional cross linker. In a second reaction step a
Cys-terminated peptide (a second binding molecule) is conjugated in
parallel together with a negative charged Cys-peptide (like
RRRLC-amid) to the maleimide group. Thereby additional negative
charged groups are placed on the metal nanoparticle surface.
[0160] FIG. 5 (B) illustrates the two step functionalization
reaction of positive charged metal nanoparticles. First aminated
metal nanoparticles are functionalized with a SulfoNHS-PEG-MAL
heterobifunctional cross linker. In a second reaction step a
Cys-terminated peptide (a second binding molecule) is conjugated in
parallel together with a positive charged Cys-peptide (like
CLDDD-OH) to the maleimide group. Thereby additional positive
charged groups are placed on the metal nanoparticle surface.
EXAMPLES OF THE INVENTION
Example 1--Functionalization of Gold Nanoparticles
[0161] The functionalization is performed by the gold (metal)-thiol
reaction using either SH-PEG-COOH heterobifunctional reagents with
a molecular weight of 400 Da. A 10 mg/ml SH-PEG-COOH (MW 634.77
g/mol) is added to 10 nM of gold nanoparticles having a diameter of
15 nm (functionalization works in the same way also for gold
nanoparticles having a diameter of 20 nm, 10 nm or 5 nm) and
incubated for 4-24 hours at RT. After the metal-thiol reaction is
completed the Au nanoparticles are washed in water and PBS. The
stability of the Au particles is determined by an UV/VIS spectral
analysis. Stable Au nanoparticles show a high absorption at 520 nm
and no absorption at 700 nm, whereas instable Au nanoparticles show
a great absorption at 700 nm and a decreased absorption at 520
nm.
Example 2--Two-Step Procedure to Conjugate a Second Binding
Molecule and a Negative Charged Gold Nanoparticle
[0162] A gold nanoparticle is functionalized with SH-PEG-COOH to
carry a negative charge. The charged gold nanoparticle shall be
conjugated to a second binding molecule (which is a peptide) via a
thiol-maleimide reaction in a two-step procedure according to the
invention.
[0163] Step 1
[0164] The functionalization is performed by a Mal-PEG-NH2
heterobifunctional reagents. In order to react the amino (NH2)
group of the heterobifunctional cross linker with the carboxyl
groups of the nanoparticle, an EDC/Sulfo NHS activation reaction is
required. Therefore 0.4 mg EDC and 1.1 mg Sulfo-NHS are added to
100 .mu.l of 10 nM gold nanoparticles for 15 min at room
temperature. After activation of carboxyl groups the reaction
mixture is purified from excess reagent by molecular weight cut-off
columns and transferred into PBS pH 7.2. Immediately after
purification a 10-100 fold molar excesses of Mal-PEG-NH2
heterobifunctional cross linker is added for 30 min at room
temperature. The excess reagents are purified off by molecular
weight cut-off columns.
[0165] Step 2
[0166] The purified nanoparticle-PEG-Mal conjugate is directly used
for the peptide conjugation reaction. The reaction takes place at a
pH between 6.5 and 7.5. Thiol-containing compounds, such as
dithiothreitol (DTT) and beta-mercaptoethanol (BME), are excluded
from reaction buffers used with maleimides because they will
compete for coupling sites.
[0167] DTT, which is used to reduce disulfides, to make sulfhydryl
groups available for conjugation is thoroughly removed using a
desalting column before initiating the maleimide reaction. Since
the disulfide-reducing agent TCEP does not contain thiols it is not
removed before reactions involving maleimide reagents. Excess
maleimides are quenched at the end of a reaction by adding free
thiols. EDTA is included in the coupling buffer to chelate stray
divalent metals that otherwise promote oxidation of sulfhydryls
(non-reactive). The conjugated peptides are added in a 10-1,000
fold molar excess. The conjugation reaction takes place at room
temperature for 2-4 hours. The excess reagents are purified off by
molecular weight cut-off columns.
Example 3--Two-Step Procedure to Conjugate a Second Binding
Molecule in Parallel Together with Additional Negative Charged
Molecules to a Negative Charged Gold Nanoparticle
[0168] A gold nanoparticle is functionalized with SH-PEG-COOH to
carry a negative charge. The charged gold nanoparticle shall be
conjugated to a second binding molecule (which is a peptide) and to
an additional negative charged molecule (which is a peptide of the
sequence: RRRLC-OH) via a thiol-maleimide reaction in a two-step
procedure according to the invention.
[0169] Step 1
[0170] The functionalization is performed by a Mal-PEG-NH2
heterobifunctional reagents. In order to react the amino (NH2)
group of the heterobifunctional cross linker with the carboxyl
groups of the nanoparticle, an EDC/Sulfo NHS activation reaction is
required. Therefore 0.4 mg EDC and 1.1 mg Sulfo-NHS are added to
100 .mu.l of 10 nM gold nanoparticles for 15 min at room
temperature. After activation of carboxyl groups the reaction
mixture is purified from excess reagent by molecular weight cut-off
columns and transferred into PBS pH 7.2. Immediately after
purification a 10-1,000 fold molar excesses of Mal-PEG-NH2
heterobifunctional cross linker is added for 30 min at room
temperature. The excess reagents are purified off by molecular
weight cut-off columns.
[0171] Step 2
[0172] The purified nanoparticle-PEG-Mal conjugate is directly used
for the peptide conjugation reaction. The reaction takes place at a
pH between 6.5 and 7.5. Thiol-containing compounds, such as
dithiothreitol (DTT) and beta-mercaptoethanol (BME), are excluded
from reaction buffers used with maleimides because they will
compete for coupling sites.
[0173] The conjugated peptides are added in a 10-1,000 fold molar
excess whereby the additional negative charged molecule has a 5-20
fold molar excess compared to the second binding molecule. This
means if the second binding molecule is used in 10 fold molar
excess, the additional negative charged molecule has a 50-200 fold
molar excess compared to the gold nanoparticle concentration. The
conjugation reaction takes place at room temperature for 2-4 hours.
The excess reagents are purified off by molecular weight cut-off
columns.
Example 4--Coupling of a First Binding Molecule on swCNTs
[0174] The single walled CNT (swCNT) network is present on a
biosensor surface and shall be functionalized with a first binding
molecule (an antibody).
[0175] First a 1 mM 1-pyrenebutric acid solution in EtOH is
incubated for 1-24 hours at room temperature. The excess reagent is
purified off by washing the sensor 3 times with EtOH followed by a
subsequent 3 times washing step with water. The functionalization
of the antibody is performed by coupling the antibody amino groups
with the carboxyl group of the 1-pyrenebutric acid. Consequently
the carboxyl groups of the 1-pyrenebutric acid have to be activated
by an EDC/Sulfo NHS activation reaction. Therefore 0.4 mg EDC and
1.1 mg Sulfo-NHS are added to 1 ml of an amino free buffer like PBS
(pH 6.0). The activation reaction takes place for 15 minutes at
room temperature.
[0176] Directly after the activation reaction the antibody is added
in a concentration of 1-0.1 mg/ml to the biosensor at pH 7.2-8.0
for 1-4 hours at room temperature. The excess antibody is purified
off by washing the sensor 3 times with PBS pH 7.2.
Example 5--Test of Sensor Functionality
[0177] The current sensor chips are conducted by crocodile clamps
to a dual-channel source meter (Keithley 2612B). The samples are
applied by a pipet. Sample volumes are varied between 10 and 50
.mu.l.
[0178] As gate electrode a Ag/AgCl electrode operating in a top
gate setting was used. A feedback circuit was also implemented,
which measures constantly the applied gate current and regulates
the gate current voltage if necessary.
[0179] In a pre-test the sensitivity to fluids with different
pH-values was tested. As result it was found that the sensor reacts
very strongly and reliably to a change between pH 6 and pH 7
solutions.
[0180] To test the measurement set up and the general sensor
functionality different pH PBS buffers were subsequently applied on
the sensor surface. Therefore, a PBS solution (pH 7) was mixed with
20 nm Au nanoparticles (Au-NP) and a concentration of 2.4 pM. The
same PBS solution without Au-NP served as reference. It could be
shown that there is a significant sensor response when the two
fluids are exchanged cyclically. This confirms that the
semiconductor sensor is influenced by low concentrations of
Au-NP.
Example 6--Measurement of the Biomarker C-Reactive Protein
(CRP)
[0181] A complex comprising a second binding molecule that is
coupled to Au nanoparticle via a linker has been produced as
described in example 1. 5 nM Au particles with 10 functionally
coupled peptides per Au particle were used in the experiment.
[0182] The biosensor was functionalized as described in example 4,
in this experiment with the monoclonal mouse IgG1 anti human CRP
antibody B08. The affinity of the antibody was first tested against
CRP (SEQ ID NO: 1) and the modified peptide sequences of SEQ ID
NOs: 2 to 6. Sequences are shown in the following table:
TABLE-US-00001 Amino acid Peptide-ID sequence SEQ ID NO: Original
sequence of CVFPKESD 1 CRP 80712 CAFPKESD 2 80713 CVFPRESD 3 80714
CVFPKDSD 4 80715 CVFPKETD 5 80716 CVYPKESD 6
[0183] The results of the affinity measurements are shown in FIG.
6. The subsequent displacement measurements were performed with the
peptide 80715 (SEQ ID NO: 5).
[0184] The Au nanoparticles are bound to the CRP-specific antibody
via a peptide and exert a field effect on the semiconductor. If the
biomarker (in this case CRP) is present in the blood sample, it can
displace the nanoparticle and thus annul the field effect.
[0185] Results of the measurement of a displacement reaction: The
current/voltage curve shows the change in the transistor property
(by annulling the field effect). First PBS without biomarker was
added, then the concentration of the biomarker CRP in PBS was
gradually increased. By adding different concentrations of the
biomarker CRP, the current-voltage curve of the transistor has
changed accordingly. The following CRP concentration s were used:
381 fM, 3 pM, 24 pM, 195 pM, 1.56 nM, 12.5 nM, 100 nM and 800 nM.
The voltage changes measured are shown in the following table:
TABLE-US-00002 CRP concentration .DELTA.V (V) 381 fM -0.009 3 pM
-0.013 24 pM -0.016 195 pM -0.017 1.56 nM -0.020 12.5 nM -0.022 100
nM -0.024 800 nM -0.027
[0186] At constant current, changes in the biomarker concentration
were measured as voltage changes. It was found that there is a
nearly linear relationship between voltage change (.DELTA.V in V)
and CRP concentration. The biomarker CRP could be reliably detected
in a concentration range between 800 nM to 381 fM. Reaction times
were 10 minutes, measurements were performed in PBS (i.e. 150 mM
salt concentration).
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Sequence CWU 1
1
618PRTHomo sapiens 1Cys Val Phe Pro Lys Glu Ser Asp1
528PRTArtificial sequenceSynthetic peptide 2Cys Ala Phe Pro Lys Glu
Ser Asp1 538PRTArtificial sequenceSynthetic peptide 3Cys Val Phe
Pro Arg Glu Ser Asp1 548PRTArtificial sequenceSynthetic peptide
4Cys Val Phe Pro Lys Asp Ser Asp1 558PRTArtificial
sequenceSynthetic peptide 5Cys Val Phe Pro Lys Glu Thr Asp1
568PRTArtificial sequenceSynthetic peptide 6Cys Val Tyr Pro Lys Glu
Ser Asp1 5
* * * * *